The Principal Investigator (PI) hypothesizes that calpain, a family of Ca++-regulated neutral cysteine proteases, regulates the activity and subcellular localization of the constitutive isoform of nitric oxide synthase (eNOS) in hypoxic lung endotheial cells (LEC). To test this hypothesis, the PI proposes a multidisciplinary approach focusing on the following four interrelated specific aims. Aim #1: to verify that calpain is responsible for loss of eNOS activity in hypoxic LEC. To address this, the PI will study the effects on eNOS activity and nitric oxide (NO) production in porcine LEC of calpain inhibition mediated by (1) pharmacologic agents, (2) ovrexpression of calpastatin, the endogenous biologic inhibitor of calpain, and (3) antisense depletion of calpain. Aim #2: To determine whether calpain affects the subcellular localization of eNOS in hypoxic LEC. To address this aim, the PI will study the effects of calpain inhibition on eNOS subcellular localization in normoxic and hypoxic porcine LEC using (1) laser scanning confocal microscopy with deconvolution capability and (2) subcellular fractionation studies. Defining factors that regulate eNOS subcellular localization will greatly advance our understanding of NO biology because subcellular localization of eNOS determines optimal NO production by endothelial cells and affects the function of NO as a messenger molecule. Aim #3: To identify the role of the actin binding proteins fodrin and heatshock protein 90 (Hsp90) in the calpain-mediated changes in eNOS in hypoxic LEC. To address this aim, the PI will examine the effects of normoxia and hypoxia in the presence and absence of calpain inhibition on Hsp9O and fodrin contents and on the protein: protein associations between Hsp90 and eNOS and fodrin and eNOS using immunoblot and co-immunoprecipitation analyses, sucrose gradient ultracentrifugation, and deconvolution microscopy. The PI will also evaluate the effects of calpain inhibition and actin stabilization on the actin-cytoskeleton architecture in normoxic and hypoxic LEC using fluorescence microscopy. Aim #4: To evaluate whether calpain inhibition prevents or attenuates the decrease in NO production and/or endothelium-dependent vasodilation in intact hypoxic pulmonary arteries. To address this aim, the PI will assess whether alpain inhibition prevents or attenuates endothelium-dependent vasorelaxation, NO production, and eNOS activity in intact porcine pulmonary arteries exposed directly to hypoxia. The results of these studies will advance our understanding of the mechanisms by which hypoxia alters pulmonary endothelial and vascular physiology in patients with lung disease and will lead to more effective care and to new and improved ways to reverse or attenuate pulmonary vascular complications such as hypertension, cor pulmonale, and impaired hypoxic vasoconstriction in these patients.